Enzymes are useful as catalysts in various reactions, and are preferably used in a purified form, separated from the organisms that produced them. In such a purified form the enzyme is relatively unstable and easily denatured, and it is also recovered with difficulty from an aqueous reaction medium. To overcome these difficulties, it is desirable to immobilize the enzyme on some insoluble carrier, where it may readily contact the reactants in the reaction medium, but where it benefits both from an increased stability and from easy recovery by simple processes such as filtration.
A conventional mechanism for immobilizing protein enzymes on a carrier is the reaction of an active hydrogen-bearing group, e.g., an alcohol, amine or mercaptan group, on the enzyme with an oxirane, or epoxy, ring pendant to an insoluble polymer, in which the ring is opened and the hydrogen from the enzyme group forms a hydroxyl group with the oxirane oxygen, as for example: ##STR1## Thus, a carrier containing a significant fraction of oxirane rings may readily immobilize protein enzymes.
Such carriers have been prepared in the past in the form of beads with diameters from about 35 .mu.m to about 2 mm, using conventional, suspension polymerization. Typical of such carriers are those described in U.S. Pat. Nos. 3,844,892 and 3,932,557. These references also teach many of the monomers containing the oxirane, or epoxy, ring.
The carriers described in the above references are conventional gel beads. As such, they have no permanent macroporosity and have a surface area approximately equal to that of spheres the same diameter as the beads. The enzymes tend to react at, and reside upon, the surface of these carriers, so increased surface area is highly desirable. Surface area of conventional, suspension-polymerized beads is typically increased by introducing macroporosity, usually by adding a phase separator, that is, a liquid which causes separation of the copolymer from the monomer phase. For such macroporosity to be useful in enzyme carriers, the pores must be large enough to permit the free passage of both the enzymes to be immobilized, and the components of the reaction medium. Methods for introducing macroporosity into the carrier beads have been reported, notably in British Pat. No. 1,512,462 and U.S. Pat. No. 4,070,348. These methods, however, produce carriers that have significant limitations for use in enzyme immobilization processes. The inverse suspension polymerization process of the U.S. reference, in which a water solution of monomers is suspended as droplets in an oil phase, produces carrier beads of relatively low surface area, while the alcohols employed as phase separators in the British reference react with the oxirane rings to reduce the number of available sites for enzyme immobilization.